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In the developing cerebral cortex, the marginal zone (MZ), consisting of early-generated neurons such as Cajal-Retzius cells, plays an important role in cell migration and lamination. There is accumulating evidence of widespread excitatory neurotransmission mediated by γ-aminobutyric acid (GABA) in the MZ. Cajal-Retzius cells express not only GABAA receptors but also α2/β subunits of glycine receptors, and exhibit glycine receptor-mediated depolarization due to high [Cl−]i. However, the physiological roles of glycine receptors and their endogenous agonists during neurotransmission in the MZ are yet to be elucidated. To address this question, we performed optical imaging from the MZ using the voltage-sensitive dye JPW1114 on tangential neocortical slices of neonatal rats. A single electrical stimulus evoked an action-potential-dependent optical signal that spread radially over the MZ. The amplitude of the signal was not affected by glutamate receptor blockers, but was suppressed by either GABAA or glycine receptor antagonists. Combined application of both antagonists nearly abolished the signal. Inhibition of Na+, K+-2Cl− cotransporter by 20 µM bumetanide reduced the signal, indicating that this transporter contributes to excitation. Analysis of the interstitial fluid obtained by microdialysis from tangential neocortical slices with high-performance liquid chromatography revealed that GABA and taurine, but not glycine or glutamate, were released in the MZ in response to the electrical stimulation. The ambient release of taurine was reduced by the addition of a voltage-sensitive Na+ channel blocker. Immunohistochemistry and immunoelectron microscopy indicated that taurine was stored both in Cajal-Retzius and non-Cajal-Retzius cells in the MZ, but was not localized in presynaptic structures. Our results suggest that activity-dependent non-synaptic release of endogenous taurine facilitates excitatory neurotransmission through activation of glycine receptors in the MZ.

In the present study, optical imaging by means of voltage-sensitive dyes as well as whole-cell patch-clamp recordings were employed to monitor neurotransmission in the MZ of neonatal rat brain slices. In addition, microdialysis followed by high-performance liquid chromatography (HPLC) analyses was performed to investigate neurotransmitter release. We demonstrated that the propagation of electrically evoked activity in the MZ is mainly mediated by GABAA and glycine receptors and that repetitive stimulation increased the extracellular GABA and taurine levels. The results suggest that endogenous taurine facilitates excitatory neurotransmission in the neonatal MZ.

Materials and Methods

All experiments were performed in accordance with EU directive 86/609/EEC for the use of animals in research, and conformed to the guidelines on the ethical use of animals for animal experimentation at Hamamatsu University School of Medicine and the Johannes Gutenberg University Mainz (approved by the Landesuntersuchungsanstalt RLP, Koblenz, Germany). All efforts were made to minimize the number of animals used and their suffering.

Preparation of Tangential Brain Slices

Tangential slices of the neocortex were prepared as described previously (Kilb and Luhmann, 2000). In brief, neonatal Wistar rats (Japan SLC, Shizuoka, Japan) at postnatal day (P) P0–P3 were deeply anesthetized by hypothermia and decapitated. The brain was quickly removed and stored for 1–2 min in ice-cold artificial cerebrospinal fluid (ACSF). The solution contained the following (mM): 220 sucrose, 2.5 KCl, 1.25 NaH2PO4, 12.0 MgSO4, 0.5 CaCl2·2H2O, 26.0 NaHCO3, 30.0 glucose. Hemispheres were dissected at the midline, the pia mater was removed, and tangential slices were cut at a maximal thickness of approximately 400 µm containing the MZ. The slices were subsequently mounted on fine tissue paper and were allowed to recover for at least 60 min in standard ACSF consisting of (mM): 126 NaCl, 2.5 KCl, 1.25 NaH2PO4, 2.0 MgSO4, 2.0 CaCl2, 26.0 NaHCO3 and 20.0 glucose. Slices were either placed in a tightly sealed box filled with 95% O2–5% CO2 at a pressure of 50 kPa at room temperature (Fukuda and Prince, 1992) or kept in an incubation chamber with ACSF saturated with 95% O2–5% CO2 at room temperature.

Optical Recording with Voltage-Sensitive dye

Tangential neocortical slices were incubated with the voltage-sensitive dye JPW1114 (100 µM) for 30 min. The paper-mounted slices were then transferred into a submerged recording chamber (volume ca. 1 mL) attached to a fixed stage of an upright microscope (E600-FN, Nikon, Tokyo, Japan) and were superfused with standard ACSF at a rate of 1–2 mL/min. A bipolar tungsten electrode (World Precision Instruments, Sarasota, FL, USA) was used to evoke synaptic transmission in the tangential neocortical slice. Synaptic responses were evoked by a current pulse of 1 mA with 100 µs duration delivered at 1 Hz. Eight consecutive responses were averaged for analysis. All experiments were performed at 30 ± 0.5°C.

A 24-V/300-W tungsten-halogen lamp was used for excitation of the voltage-sensitive dye. After slices were viewed with a 4× objective lens (N. A. 0.10; Nikon), optical recording was achieved by using a 10× water immersion objective lens (N.A. 0.30; Nikon) and filter cube comprised of a 510–560 nm excitation filter, a 570 nm dichroic mirror, and a 590 nm barrier filter. Emitted fluorescence was detected by a high-speed 80 × 80 pixel back-thinned charge-coupled device (CCD) camera (NeuroCCD, Red Shirt Imaging, Fairfield, CT, USA). Photocurrents were converted to voltages and were DC-coupled to analog-to-digital converters using a 16-bit resolution at a frame rate of 1 kHz. Off-line data analysis was performed using the NeuroPlex software (Red Shirt Imaging).

Dye bleaching was corrected off-line and light intensities were measured as relative fluorescence change (ΔF/F), where F is the fluorescent light intensity of the stained slice during illumination without evoked neuronal activity and ΔF is the fluorescence change during neuronal activity. A decrease in fluorescence (plotted upwards in all figures) corresponded to membrane depolarization, and an increase in fluorescence plotted downwards corresponded to membrane hyperpolarization.

Microdialysis-HPLC Measurement

A probe for microdialysis was inserted into the tangential neocortical slice at approximately 800 µm apart from the stimulating electrode. The microdialysis collections started more than 60 min before stimulation began. The procedure for microdialysis was described previously (Nakahara et al., 2001). In brief, the dialysis probe was perfused with the standard ACSF at a flow rate of 2 µL/min. Measurements of steady-state levels of amino acids began after a 60-min stabilization period. Three consecutive samples were collected at 20-min intervals in small plastic vials to determine steady-state levels, followed by six consecutive samples to establish temporal changes induced by electrical stimulation. After samples were derivatized with o-phthalaldehyde, the concentrations of amino acid neurotransmitters including GABA, glutamate, glycine and taurine were determined by reverse-phase HPLC with electrochemical detection. The HPLC system (BAC-300 system, EICOM, Kyoto, Japan) consisted of an EP-300 pump, an ECD-300 electrode, and a PowerChrom system (ADI, Sydney, Australia). Separation of amino acid derivatives was achieved using an Eicompack MA-5ODS column. The detection potential was set at +700 mV against an Ag/AgCl reference electrode. The flow rate was 1.20 mL/min, and the sensitivity was set at 64 nA/V full-scale. The mobile phase consisted of 100 mM phosphate buffer (pH 6.0) and 30% (v/v) methanol. The amino acids and the derivatives were mixed and allowed to react for exactly 2 min before injection.

Immunohistochemistry

Animals were perfusion-fixed with 4% (w/v) paraformaldehyde and 0.5% (v/v) glutaraldehyde in 0.1 M phosphate-buffered saline (PBS) and brains removed were post-fixed with the same fixative. The sections were cut at 20 µm on a cryostat, thaw-mounted onto silane-coated slides. The sections were pretreated with 0.5% (w/v) sodium borohydride for 30 min. Sections were first treated in 0.3% (v/v) H2O2 for 30 min. After washing with PBS, the sections were incubated with a blocking solution (10% (v/v) normal goat serum, 0.1% (v/v) Triton X-100 in PBS) for 1 h at room temperature. Sections were then incubated at 4°C for 36 h with rabbit anti-taurine polyclonal antibody (TT100; 1:100; Signature Immunologics, Salt Lake City, UT, USA), for 1 h with biotinylated goat anti-rabbit IgG (1:100; Vector Laboratories, Burlingame, CA, USA). After rinsing in 0.1 M PBS, the sections were incubated with avidin–horseradish peroxidase (Vectastain ABC kit; Vector Laboratories) for 30 min at room temperature. After washing with PBS, the immunoreaction was developed for 2–3 min with 0.01% (w/v) 3,3′-diaminobenzidine (Sigma-Aldrich, St. Louis, MO, USA) activated by 0.01% (v/v) H2O2 .

Electron Microscopy

After the animals (P0) were deeply anesthetized with halothane and perfused transcardially with 4% (w/v) paraformaldehyde/0.5% (v/v) glutaraldehyde in 0.1 M HEPES buffer, the brains were removed and post fixed for 10 min. Coronal sections were cut at 50 µm with a vibratome (DTK-1500; Dosaka, Kyoto, Japan) in the same fixative solution. Sections were kept in 0.1 M HEPES buffer for 1 day at 4°C. After rinsing in PBS, sections were incubated for 36 h at 4°C with polyclonal antibody against taurine (Signature Immunologics). The sections were washed with PBS several times, and then treated with anti-rabbit secondary antibody coupled with 1.4-nm-diameter gold particles (Nanogold; Nanoprobes, Yaphank, NY, USA) overnight at room temperature. Immunoelectron microscopic analysis was performed by Tokai-EMA (Nagoya, Japan). After the sections were washed with PBS and fixed in 2% (v/v) glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) for 3 h at room temperature, gold particles were enlarged for microscopic examination with the HQ-Silver Enhancement Kit (Nanoprobes) and routinely processed for electron microscopic examination. The embedded samples were sectioned (thickness 80 nm) with an ultra-microtome (LKB2088; LKB, Bromma, Sweden) and stained with uranyl acetate and lead citrate. Sections were then carbon-coated in a vacuum and observed with a JEOL transmission electron microscope (JEM 2000EX; JEOL, Tokyo, Japan) at 100 kV.

Statistics

Unless otherwise indicated, all numerical data are shown as the mean ± the standard error of the mean (SEM). Differences between two groups were assessed using Student’ s paired t-test for absolute and Mann–Whitney U-test for normalized values. Comparisons among several groups were performed using one-way analysis of variance (ANOVA) with post-hoc Tukey test. In the microdialysis study, the Friedman test was used to evaluate the overall response with time. A P < 0.05 was considered statistically significant.

Results

Because excitatory GABAergic neurotransmission was reported in the developing MZ (Mienville, 1998; Schwartz et al., 1998; Aguiló et al., 1999; Dammerman et al., 2000a; Kilb and Luhmann, 2001; Soda et al., 2003; Achilles et al., 2007; Kirmse et al., 2007), we hypothesized that excitatory propagation mediated by the GABAergic system could be detected along the tangential direction of the MZ (Figure 1A). Optical signals from the MZ in tangential neocortical slices stained with the fluorescent voltage-sensitive dye JPW1114 were monitored with a high-speed CCD camera. Potentials evoked by single electrical stimulations on the edge of the recorded area spread out rapidly over the MZ monitored (Figure 1B). The spread of excitation was also apparent by spatiotemporal recordings of optical signals (−ΔF/F) in each slice, of which representative traces were analyzed (Figure 1C).

FIGURE 1

Figure 1. Optical voltage recording in tangential neocortical slices. (A) Schematic diagram showing cellular and axonal components of the tangential direction of the MZ. (B) Left: schematic diagram showing preparation of tangential slice and optical recording from the MZ. Right: the main component of the optical signal evoked by single electrical stimulations spread out over the MZ, so that excitatory optical signals were recorded spatially (colored in red). Note that the stimulating electrode was placed on the edge of the recorded area (see panel at 0 ms). (C) Spatiotemporal recordings enable us to analyze the evoked optical signals which spread in horizontal direction within the MZ. Representative traces were chosen (inset) for analyses at distances of 800 µm from the stimulation sites.

When the glycine receptor antagonist STR (50 µM) was added to ACSF containing CNQX and D-AP5, the peak optical signal values were significantly reduced (56.4 ± 0.5% CNQX/D-AP5, n = 7, P < 0.001, paired t-test; Figure 2D), suggesting that glycine receptors were also involved in the propagation of evoked signals. Thus, the spread of excitation in the MZ was mediated not only by GABAA receptors, but also by glycine receptors.

To determine whether GABAA and glycine receptors are differentially activated by endogenous agonists, we examined the additive effects of both antagonists. Figure 2E shows that PTX partially suppressed the evoked optical signals, and that a subsequent addition of STR further suppressed the signals, indicating that both GABAA and glycine receptors were activated. Interestingly, the evoked optical signals were almost completely suppressed by the combined application of PTX and STR even in the absence of CNQX and D-AP5 (14.1 ± 1.2% control, n = 4, P < 0.001, paired t-test; Figure 2E), indicating that co-activation of GABAA and glycine receptors, but not glutamate receptors, should be responsible for the synaptic propagation of evoked optical signals in the MZ. These results suggest that the spread of the optical signals evoked by electrical stimulation in the MZ is attributable to evoked release of GABAergic and glycinergic agonists.

Electrophysiological responses of single Cajal-Retzius cells to electrical stimulation: (A) Voltage traces illustrating the typical response of a Cajal-Retzius cell upon injection of de- and hyper-polarizing current pulses. (B) Five consecutive current-traces illustrating typical inward current evoked by electrical stimulation in a tangential slice. (C) The stimulus-evoked inward current was abolished in the presence of 1 µM TTX (magenta trace). (D) Two typical current traces recorded from the same trial illustrating that the stimulus-induced inward current is unaffected in the presence of 50 µM D-AP5 and 10 µM CNQX (purple trace). (E) The stimulus-induced inward current was reduced in the presence of 1 µM STR (green trace) and was abolished when both GABAA and glycine receptors were blocked with 50 µM STR and 50 µM PTX, respectively (red trace). (F) The stimulus-induced inward current was reduced in the presence of 50 µM PTX (blue trace). (G) Statistical analysis. Bars represent the mean ± SEM, numbers of experiments are given in the bars.

Evoked Release of GABA and Taurine by Focal Electrical Stimulation

To examine whether focal electrical stimulation that caused the spread of excitation over the MZ could affect release of endogenous agonists of GABAA and glycine receptors, we monitored the release of GABA, glycine, and taurine besides glutamate from the tangential neocortical slices containing the MZ using microdialysis followed by HPLC. The results are shown in Figure 4. The extracellular concentration of GABA significantly increased from the baseline (1.07 ± 0.07 pmol/40 µL, n = 7) to the peak (1.45 ± 0.41 pmol/40 µL, 167.8 ± 43.4% baseline) and then returned to the baseline (P = 0.012, Friedman test; Figure 4A). The peak % baseline value of extracellular GABA concentration was significantly greater than that of the control (insertion of dialysis probe without stimulation) (P = 0.024; Mann–Whitney U-test). By contrast, the extracellular concentration of glutamate (3.87 ± 0.17 pmol/40 µL; n = 5) did not increase during stimulation (3.37 ± 0.96 pmol/40 µL, P = 0.691, Mann–Whitney U-test; Figure 4B), confirming that excitatory synaptic transmission within the MZ is partially mediated by GABA but not by glutamate (Dammerman et al., 2000a).

FIGURE 4

Figure 4. Temporal changes in neuro-active amino acid concentrations after focal electrical stimulation assessed by microdialysis followed by HPLC. Evoked release (red) of GABA (A) glutamate (B) glycine (C) and taurine (D) were estimated and are indicated as % baseline value. In controls (black), no stimulation was applied. Time after the stimulation is indicated in abscissa (min). Electrical stimulation of 1 mA for 100 µs was delivered at 1 Hz for 20 min, the periods of which are indicated by bars. Baseline values were defined as the average value 60 min immediately before stimulation. Note that taurine and GABA (with 1 mM nipecotic acid), but neither glycine nor glutamate, were significantly increased in response to the electrical stimulation in the MZ. ** P < 0.01, * P < 0.05 as compared with control (Mann–Whitney U-test).

Figure 5. TTX reduced ambient taurine levels. Temporal changes in extracellular taurine concentrations after bath application of 1 µM TTX for 60 min during microdialysis were estimated and are indicated as % baseline value. TTX was applied as indicated. The baseline value was defined as the average value 60 min immediately before TTX application and the control data are from Figure 4D. *P < 0.05 as compared with control (Mann–Whitney U-test).

Cellular and Subcellular Localization of Taurine

Localization of taurine in the MZ of the developing cerebral cortex was examined by immunohistochemistry. At P0, immunoreactivity for taurine was found in both Cajal-Retzius-like and non-Cajal-Retzius-like cells (Figure 6A). These results suggested that both cells in the MZ could be a source of taurine. To further identify the precise localization of taurine in these cells, we performed immuno-electron microscopic analysis with a taurine antibody. Interestingly, electron dense signals for taurine immunoreactivity were present intracellularly (Figure 6B), although these were not observed in presynaptic structures such as synaptic vesicles in the specimens that we examined. These results suggested that taurine may be released from the cell soma.

Taurine transporter did not contribute to the spread of excitation

It is known that external taurine stimulates taurine uptake by the Na+-dependent taurine transporter, inducing a membrane depolarization by the net movement of positive charge with the stoichiometric transport of 2 Na+: 1 Cl−: 1 taurine (Sarkar et al., 2003) and a long-lasting enhancement of neurotransmission (Chepkova et al., 2002; Sergeeva et al., 2003; Chepkova et al., 2006). To assess the role of taurine transporter activity in the spread of excitation in the MZ, we studied the effects of a competitive taurine transporter inhibitor, GES, on the propagation of evoked signals. The application of GES (1 mM) did not affect evoked optical signals (97.1 ± 3.9%, n = 4, P = 0.063, paired t-test; Figure 7). Thus, GES should have no influence on the spread of excitation in the MZ.

FIGURE 7

Figure 7. Propagation of excitation was not affected by the taurine transporter inhibitor GES. Typical traces of evoked optical signals with or without GES. Note that application of GES (1 mM) did not affect the amplitude of the signals (red trace). GES was applied at least for 10 min before stimulation.

Discussion

In the present study, we demonstrated that excitatory neurotransmission along the tangential axis of the MZ is mediated mainly by GABAA and glycine receptors but not by glutamate receptors. In addition, we showed that GABA and the GABAergic and glycinergic agonist taurine, but not glycine or glutamate, are released upon electrical stimulation. We conclude from these observations that GABA and taurine may act as endogenous agonists of both GABAA and glycine receptors (Flint et al., 1998; Kilb et al., 2002), thereby facilitating excitatory neurotransmission in the neonatal MZ.

Our present study demonstrates that TTX-sensitive neuronal activity is required for both excitatory propagation and ambient taurine release in the neonatal MZ. In contrast to the complete blockade of postsynaptic currents in the patch-clamp experiments, in optical voltage recordings a Ca2+-insensitive component remains, which most probably reflects stimulus-induced depolarization of presynaptic elements within the MZ. Because propagation depends on neurotransmission mediated by activation of glycine receptors as well as GABAA receptors, evoked and/or activity dependent release of taurine, an endogenous agonist preferentially for glycine receptors, should play a key role in the propagation of excitation over the MZ. It has already been reported that taurine can tonically activate glycine receptors in migrating and differentiating neurons in the immature cortical plate before synaptogenesis occurs (Flint et al., 1998). Under these conditions, taurine is released in the absence of action potentials and extracellular calcium. In addition, a non-synaptic taurine release has also been suggested in the MZ of the developing neocortex (Kilb et al., 2008). Although we did not investigate the release mechanism of neuronal activity-dependent ambient taurine, our present findings shows that evoked taurine release is involved in neurotransmission in the MZ. While it is shown that taurine can be released from the immature cerebral cortex in response to depolarization or tetanic electrical stimulation (Collins and Topiwala, 1974; Oja and Saransaari, 2013), the mechanism underlying activity-dependent release of taurine remains unclear (Collins and Topiwala, 1974; Oja and Saransaari, 2013).

Previously we reported glycine receptor mediated depolarization (Kilb et al., 2002) and the functional expression of α2/β glycine receptors (Okabe et al., 2004) in rat Cajal-Retzius cells. A recent study suggests that glycine receptors are also involved in corticogenesis by controlling radial neuronal migration (Nimmervoll et al., 2011). However, it is so far unknown whether glycine receptor-mediated depolarization affects neurotransmission. Our present results suggest that they facilitate neurotransmission, which spreads over the MZ. The effects of the GABAA and glycine receptor antagonists were additive, this result suggests that not only GABAA receptors but also glycine receptors are involved in synaptic transmission in the MZ. The mechanism underlying neurotransmission mediated by glycine receptors could be different from that reported recently in the developing visual cortex, as tonic activation of presynaptic glycine receptors enhanced excitatory neurotransmitter release (Kunz et al., 2012).

Taurine has been shown to act as an endogenous agonist for glycine receptors, with a lower affinity than glycine (Flint et al., 1998; Kilb et al., 2002; Okabe et al., 2004; Le-Corronc et al., 2011). The results from microdialysis suggest that an endogenous agonist for glycine receptors should be taurine but not glycine, because taurine was released by electrical stimulation. In addition, immunohistochemistry for taurine revealed that both Cajal-Retzius-like cells and non-Cajal-Retzius-like cells contain taurine. Moreover our immunoelectron microscopic analysis revealed that taurine was present inside the cell soma, but not in synaptic structures (Figure 6). It has been reported that taurine activates not only glycine receptors but also GABAA receptors (Chepkova et al., 2002; Jia et al., 2008; Le-Corronc et al., 2011). Furthermore, taurine can modulate GABAA-receptor mediated neurotransmission (Sergeeva et al., 2007). Thus, taurine released by stimulation might activate and/or modulate GABAA receptors as well as glycine receptors.

Several reports demonstrated that taurine induces long-lasting enhancement of neurotransmission. In corticostriatal pathway, an involvement of taurine uptake by Na+-dependent taurine transporters accompanied by membrane depolarization has been considered (Chepkova et al., 2002; Sarkar et al., 2003; Sergeeva et al., 2003; Chepkova et al., 2006). Although such a mechanism might also facilitate the spread of excitation in the MZ, it is unlikely according to our present results, because the taurine transporter inhibitor GES had no effect on the propagation of the evoked signals. By contrast, in the hippocampus, a robust long lasting potentiation of synaptic transmission by taurine is dependent on GES-sensitive taurine transport (Galarreta et al., 1996; del Olmo et al., 2004; Dominy et al., 2004). In the present study, GES did not affect the electrical stimulation-induced excitatory propagation in the MZ; despite that, inhibiting taurine uptake can increase extracellular taurine levels. One possible explanation is that extracellular taurine increased by electrical stimulation might already reach a saturating level of facilitation in the propagation of excitation. Although the precise mechanism underlying taurine-mediated neurotransmission remains to be identified, we suggest that taurine should facilitate excitatory propagation independent of GES-mediated depolarization.

Our immunoelectron microscopic analysis demonstrated that taurine is located inside immature neurons, but was unable to detect taurine in presynaptic structures. These results imply that release of taurine may be controlled via a non-vesicular process, which is consistent with previous studies that suggest taurine can be released as osmolyte by some non-vesicular systems in neurons or glial cells (Flint et al., 1998; Mulligan and MacVicar, 2006). Because a reverse mode of taurine transport is not likely attributable in the present study, taurine might be released by anion channels or another unidentified mechanisms that are regulated by neuronal activity. The volume-sensitive anion channel is known to be activated by cellular swelling (Inoue and Okada, 2007) as well as radical oxygen species (Liu et al., 2009), both of which could be activated by neuronal activities (Takagi et al., 2002; Kann and Kovács, 2007).

In the present study, electrical stimulation-induced elevation of extracellular taurine levels persisted for longer time than that of GABA. It could be because ambient taurine increased 100 times more than GABA, consuming more time for diffusion. Alternatively, GABA is vesicularly released in the synapse and is rapidly cleared by presynaptic and perisynapse-astrocytic GABA transporters, GAT1 and GAT3, respectively (Egawa et al., 2013), whereas taurine may be released from volume-sensitive anion channels (Ando et al., 2012; Furukawa et al., 2014) and be uptaken by taurine transporter-mediated mechanism (Galarreta et al., 1996; del Olmo et al., 2004; Dominy et al., 2004). Also as discussed above, taurine is localized in the cytoplasm, not in the presynaptic structures. Considering these differences in spatial release-uptake mechanism between GABA and taurine, clearance of taurine may need much longer time than that of GABA (see Figure 4).

In conclusion, this is the first report demonstrating that endogenous taurine is functionally involved in excitatory synaptic transmission spreading horizontally within the MZ. Taurine rather than glycine could be the endogenous agonist for glycine receptors. Thus, endogenous taurine acting on glycine receptors contribute to excitatory neurotransmission that is mediated by GABA in the MZ, in which cells have higher [Cl−]i than that expected for passive distribution. Therefore, endogenous taurine in the MZ directly influences information processing in the immature MZ, thereby possibly influencing important developmental processes, such as cell migration, axonal growth and lamination of the developing cerebral cortex.

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgments

This work was supported by Grants-in-Aid for Scientific Research on Priority Areas (#21026013) and Innovative Areas (#23115506), from the Ministry of Education, Culture, Sports, Science and Technology, Japan (to Atsuo Fukuda), and Grants-in-Aid for Scientific Research (B) #22390041, #25293052 and for Challenging Exploratory Research #23659535, #24659508 from the Japan Society for the Promotion of Science (to Atsuo Fukuda), and by DFG grants to Werner Kilb and Heiko J. Luhmann.